Introduction: Protein expression, purification, and characterization is a process where a desired protein is extracted from an organism, purified so that only the desired components of the protein are taken, and inserted within a new organism [1], implementing a new protein that would otherwise not be created naturally. The most common technique includes creating complementary DNA (cDNA), and after determining the N and C termini, the desired genes are cloned or amplified, using techniques such as Polymerase Chain Reaction (PCR). To express the protein however, a recombinant host has to be used; because of the simplicity of the organism, bacteria, such as E. Coli, are often used a recombinant host [2], allowing for the creation and growth of the desired protein. During this stage, affinity tags are also bound to the desired protein, such as histidine tags, so that the protein can effectively be traced and extracted. The protein is then further purified using high-powered liquid (gravity flow) chromatography procedures, and characterized, reducing the risk of wasting resources on protein material of inadequate quality, done through UV absorption spectroscopy (characterized with by SDS-PAGE) [3]. Thus, the hypothesis states that through the hexa-histidine tag, successful protein extraction and purification will be demonstrated through purple pellet coloration.
Materials & Methods:
25ul of the bacteria E.Coli BL21(DE3) was added into a DNA and control transformation tube. 2ul of plasmid was added to the DNA tube, and the tubes were heat shocked and placed on ice. 200ul of SOC media was added, 50ul of the bacteria mixture was placed from each tube to the DNA and control culture plates, and the plates were coilrolled. Ampicillin and a bacterial colony were added to 2 tubes of 5ml LB and stored in a shaking incubator. 25ml of LB was added to a 125ml Erlenmeyer flask with ampicillin with 0.625 ml of the starter culture. The flask was placed in a shaking incubator. 500ul of the culture was placed in an Eppendorf tube and labeled sample 1 (not necessary). The bacteria was poured into a 50ml conical tube and centrifuged. The pellet was saved, 2.5ml of 1x PBS and lysozyme were added to the tube and vortexed stored (vortex stored?). 2ul of Cyanase was added to the 50ml conical and placed on ice; the lysate was added to 1.7ml microcentrifuge tubes and centrifuged for. 50ul of the supernatant was place in a microcentrifuge tube, labeled sample 2, and stored. The lysate was syringe filtered through a 0.45ul SFCA filter. 0.5ml of Ni-NTA resin/buffer mix was added to the lysate and incubated at room temp. The lysate was placed in a 20ml Bio-Rad chromatography Ecno column and settled. The liquid was drained into a waste tube. 50ul of the waste was saved and labeled sample 3 and stored. Then, the wash buffer was flushed through, settled for 5mins, and drained; 50ul was saved, labeled, sample 4, and stored. The same process occurred with Elution 1 and Elution 2, and those 50ul samples were saved as sample 5 and 6. 10 cv of water, 10 cv of 0.5 NaOH, and 10 cv water were drained through to strip the Ni-NTA. Sample 1 was then centrifuged, and only the pellet was saved and resuspended with 200ul of water and 40ul of loading buffer. Loading buffer was also added to samples 2-6, placed in a heat block and centrifuged. Each sample and a molecular standard were run through gel electrophoresis, the gel was removed and washed, stained, and washed again to make the bands visible. The gel was dried in the Biotech lab and labeled. (Be more specific with the details of gel electrophoresis)
Results: Figure 1. Ampicillin agar plate containing only bacteria found on the surface of an iPhone after overnight incubation at 37 degrees Celsius; did not contain BL21(DE3) or pGEM-gbr22
Figure 2. Ampicillin agar plate containing BL21(DE3) and pGEM-gbr22 after overnight incubation at 37 degrees Celsius
Figure 3. Ampicillin agar control plate with no BL21(DE3) (no plasmid, NOT no cells)after overnight incubation at 37 degrees Celsius
Figure 4. BL21(DE3) bacterial cells in the log growth phase after being transformed with pGEM-gbr22 after 24 hours in the shaking incubator at 37 degrees Celsius and 250 rpm
Figure 5. BL21(DE3) bacterial cell pellet after centrifuging the bacterial cells with pGEM-gbr22 for 10 minutes at 4 degrees Celsius at 5,000 rpm; the pellet had a weight of 0.44 grams
Figure 6. Nanodrop spectrophotometer graph of 2ulof elution 2 at absorbance 280nm (A280)
Figure 6. Nanodrop spectrophotometer graph of 2ulof elution 2 (This is elution 2??) at absorbance 280nm (A280)
Figure7. Gel bands after gel electrophoresis and staining (explain the bands!!!)
Figure 8. Elution 1 tube after draining the protein through the Bio-Rad Econo chromatography column (Be more detailed)
Figure 9. Elution 2 tube after draining the protein through the Bio-Rad Econo chromatography column (Be more detailed)
Figure 10. Information chart regarding the Thermo Scientific Prestained Protein Ladder Molecular Weight (There is a better image online) Discussion: Through the process of extraction (expression), purification, and characterization, the desired protein was extracted (expressed) and ready to insert into a new organism. However, errors could have arisen during the experiment. While transforming the competent bacterial cells, the work could have been done too far from the site of the actual gas burner, allowing for contamination. Additionally, the times concerning heat shocks and blocks are significant; a minute longer could have altered results by denaturing the protein (heat block = you want to denature the protein; heat shock has to do with DNA and bacterial cell membrane permeability). When working with the various enzymes such as cyanase and lysozyme, the enzymes were not always consistently place on ice, thus allowing for the enzyme to denature. During the experiment, Lysozyme was used to digest the cell’s walls, opening the E. coli cells that express the purple protein. Cyanase was used as a nuclease to reduce the viscosity by digesting the DNA/RNA in the mixture. Concerning the HIS tag system, the overexpressed purple protein [gbr22] was modified to have six histidine residues added to the C-terminate these residues bind to divalent nickel cations that are immobilized on Ni-NTA agarose. To release the protein from the Ni-NTA agarose, imidazole is added, competing with the histidine residues for binding. When analyzing each sample, sample 1 contained the bacterial cells before harvesting the culture, sample 2 contained the lysate after centrifugation, sample 3 contained the waste, sample 4 contained the wash, and sample 5 and 6 contained elutions 1 and 2, respectively. The wash step removed the proteins loosely bound to the resin. However, the elution buffer releases the gbr22 protein from Ni-NTA resin due to its high concentration imidazole. After the gel, the protein was considerably smaller than the protein before purification because before purification, the protein was still associated with other components of the cell. (The protein was always the same size...) Conclusions: Through this process, a protein needed for ampicillin resistance (Doesn't really make sense) originally found in E. Coli was successfully extracted, amplified in a plasmid pGEM-gbr22, traced using a hexa-histidine tag colored purple (The his tag was not colored purple), purified, and analyzed for its molecular weight and ready for insertion into a new organism (We are not inserting this protein into another organism). Future Directions of this lab include extracting proteins not found in humans that are found in organisms, amplifying them, purifying, and inserting them back into the human genome (Why would we want to put these in the human genome?), providing proteins and services that humans could not produce naturally. If a certain enzyme, such as DHFR for example, which helps regulate cell growth and proliferation, malfunctioned in cancer cells within a human, those cells could be eliminated, and properly working DHFR enzymes in other organisms could be extracted, purified, and used within human cells. References:
Büssow, K. et al. Structural genomics of human proteins-target selection and generation of a public catalogue of expression clones. Microb. Cell Fact.4, 21 (2005).
Bartlam, M., Xu, Y. & Rao, Z. Structural proteomics of the SARS coronavirus: a model response to emerging infectious diseases. J. Struct. Funct. Genomics8, 85–97 (2007).
Acton, T.B. et al. Robotic cloning and Protein Production Platform of the Northeast Structural Genomics Consortium. Methods Enzymol.394, 210–243 (2005).
Title:
Show me the Protein!
Introduction:
Protein expression, purification, and characterization is a process where a desired protein is extracted from an organism, purified so that only the desired components of the protein are taken, and inserted within a new organism [1], implementing a new protein that would otherwise not be created naturally. The most common technique includes creating complementary DNA (cDNA), and after determining the N and C termini, the desired genes are cloned or amplified, using techniques such as Polymerase Chain Reaction (PCR). To express the protein however, a recombinant host has to be used; because of the simplicity of the organism, bacteria, such as E. Coli, are often used a recombinant host [2], allowing for the creation and growth of the desired protein. During this stage, affinity tags are also bound to the desired protein, such as histidine tags, so that the protein can effectively be traced and extracted. The protein is then further purified using high-powered liquid (gravity flow) chromatography procedures, and characterized, reducing the risk of wasting resources on protein material of inadequate quality, done through UV absorption spectroscopy (characterized with by SDS-PAGE) [3]. Thus, the hypothesis states that through the hexa-histidine tag, successful protein extraction and purification will be demonstrated through purple pellet coloration.
Materials & Methods:
25ul of the bacteria E.Coli BL21(DE3) was added into a DNA and control transformation tube. 2ul of plasmid was added to the DNA tube, and the tubes were heat shocked and placed on ice. 200ul of SOC media was added, 50ul of the bacteria mixture was placed from each tube to the DNA and control culture plates, and the plates were coilrolled. Ampicillin and a bacterial colony were added to 2 tubes of 5ml LB and stored in a shaking incubator. 25ml of LB was added to a 125ml Erlenmeyer flask with ampicillin with 0.625 ml of the starter culture. The flask was placed in a shaking incubator. 500ul of the culture was placed in an Eppendorf tube and labeled sample 1 (not necessary). The bacteria was poured into a 50ml conical tube and centrifuged. The pellet was saved, 2.5ml of 1x PBS and lysozyme were added to the tube and vortexed stored (vortex stored?). 2ul of Cyanase was added to the 50ml conical and placed on ice; the lysate was added to 1.7ml microcentrifuge tubes and centrifuged for. 50ul of the supernatant was place in a microcentrifuge tube, labeled sample 2, and stored. The lysate was syringe filtered through a 0.45ul SFCA filter. 0.5ml of Ni-NTA resin/buffer mix was added to the lysate and incubated at room temp. The lysate was placed in a 20ml Bio-Rad chromatography Ecno column and settled. The liquid was drained into a waste tube. 50ul of the waste was saved and labeled sample 3 and stored. Then, the wash buffer was flushed through, settled for 5mins, and drained; 50ul was saved, labeled, sample 4, and stored. The same process occurred with Elution 1 and Elution 2, and those 50ul samples were saved as sample 5 and 6. 10 cv of water, 10 cv of 0.5 NaOH, and 10 cv water were drained through to strip the Ni-NTA. Sample 1 was then centrifuged, and only the pellet was saved and resuspended with 200ul of water and 40ul of loading buffer. Loading buffer was also added to samples 2-6, placed in a heat block and centrifuged. Each sample and a molecular standard were run through gel electrophoresis, the gel was removed and washed, stained, and washed again to make the bands visible. The gel was dried in the Biotech lab and labeled. (Be more specific with the details of gel electrophoresis)
Results:
Figure 1. Ampicillin agar plate containing only bacteria found on the surface of an iPhone after overnight incubation at 37 degrees Celsius; did not contain BL21(DE3) or pGEM-gbr22
Figure 2. Ampicillin agar plate containing BL21(DE3) and pGEM-gbr22 after overnight incubation at 37 degrees Celsius
Figure 3. Ampicillin agar control plate with no BL21(DE3) (no plasmid, NOT no cells)after overnight incubation at 37 degrees Celsius
Figure 4. BL21(DE3) bacterial cells in the log growth phase after being transformed with pGEM-gbr22 after 24 hours in the shaking incubator at 37 degrees Celsius and 250 rpm
Figure 5. BL21(DE3) bacterial cell pellet after centrifuging the bacterial cells with pGEM-gbr22 for 10 minutes at 4 degrees Celsius at 5,000 rpm; the pellet had a weight of 0.44 grams
Figure 6. Nanodrop spectrophotometer graph of 2ulof elution 2 (This is elution 2??) at absorbance 280nm (A280)
Figure7. Gel bands after gel electrophoresis and staining (explain the bands!!!)
Figure 8. Elution 1 tube after draining the protein through the Bio-Rad Econo chromatography column (Be more detailed)
Figure 9. Elution 2 tube after draining the protein through the Bio-Rad Econo chromatography column
(Be more detailed)
Figure 10. Information chart regarding the Thermo Scientific Prestained Protein Ladder Molecular Weight (There is a better image online)
Discussion:
Through the process of extraction (expression), purification, and characterization, the desired protein was extracted (expressed) and ready to insert into a new organism. However, errors could have arisen during the experiment. While transforming the competent bacterial cells, the work could have been done too far from the site of the actual gas burner, allowing for contamination. Additionally, the times concerning heat shocks and blocks are significant; a minute longer could have altered results by denaturing the protein (heat block = you want to denature the protein; heat shock has to do with DNA and bacterial cell membrane permeability). When working with the various enzymes such as cyanase and lysozyme, the enzymes were not always consistently place on ice, thus allowing for the enzyme to denature. During the experiment, Lysozyme was used to digest the cell’s walls, opening the E. coli cells that express the purple protein. Cyanase was used as a nuclease to reduce the viscosity by digesting the DNA/RNA in the mixture. Concerning the HIS tag system, the overexpressed purple protein [gbr22] was modified to have six histidine residues added to the C-terminate these residues bind to divalent nickel cations that are immobilized on Ni-NTA agarose. To release the protein from the Ni-NTA agarose, imidazole is added, competing with the histidine residues for binding. When analyzing each sample, sample 1 contained the bacterial cells before harvesting the culture, sample 2 contained the lysate after centrifugation, sample 3 contained the waste, sample 4 contained the wash, and sample 5 and 6 contained elutions 1 and 2, respectively. The wash step removed the proteins loosely bound to the resin. However, the elution buffer releases the gbr22 protein from Ni-NTA resin due to its high concentration imidazole. After the gel, the protein was considerably smaller than the protein before purification because before purification, the protein was still associated with other components of the cell. (The protein was always the same size...)
Conclusions:
Through this process, a protein needed for ampicillin resistance (Doesn't really make sense) originally found in E. Coli was successfully extracted, amplified in a plasmid pGEM-gbr22, traced using a hexa-histidine tag colored purple (The his tag was not colored purple), purified, and analyzed for its molecular weight and ready for insertion into a new organism (We are not inserting this protein into another organism). Future Directions of this lab include extracting proteins not found in humans that are found in organisms, amplifying them, purifying, and inserting them back into the human genome (Why would we want to put these in the human genome?), providing proteins and services that humans could not produce naturally. If a certain enzyme, such as DHFR for example, which helps regulate cell growth and proliferation, malfunctioned in cancer cells within a human, those cells could be eliminated, and properly working DHFR enzymes in other organisms could be extracted, purified, and used within human cells.
References: